The long-term objective of this research program is to determine the neuroendocrine mechanisms regulating LH and prolactin secretion in mammals. Proposed studies examine hypothalamic mechanisms by which testosterone (T) and estradiol-17beta (E(2)) plus progesterone (P(4)) may exert their negative feedback action on tonic LH secretion in adult male and female rats. While the concept of steroid negative feedback was first posited in the 1930's, the identity of the neurons acted upon by negative feedback steroids and their relationship to the GnRH neurons are not known with certainty. Our current focus is to determine the physiological role of steroid-addressed GABAergic neurons situated in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis [DBB(ovlt)], the dorsomedial aspect of the medial preoptic nucleus (MPNdm), and the tuberoinfundibular GABAergic (TIGA) neurons, in the regulation of LH secretion. These GABAergic neurons are known to concentrate sex steroids or to express sex steroid receptor, GABAergic neurons are known to synapse on GnRH neurons, and GnRH neurons in situ recently have been shown to express GABA(A)beta(3) receptor subunit mRNA. The principal working hypothesis under investigation is that steroid- sensitive rostral hypothalamic and tuberoinfundibular GABAergic neurons mediate the negative feedback effects of gonadal steroids on GnRH neurons. Proposed research is focused on four specific aims to determine: (1) whether ovariectomy decreases preoptic and tuberoinfundibular GABAergic neurotransmission which is prevented by physiological, negative feedback concentrations of E2 + P(4), (2) whether microinfusions of steroid receptor antagonists or antisense oligonucleotides to steroid receptor mRNAs block the effects of sex steroids on GABA turnover and GAD(65 or 67) mRNA expression in these steroid-sensitive brain regions, (3) whether steroid receptor antagonist microinfusions into these steroid-sensitive brain regions increase GnRH release which is prevented by co-infusion of GABA and GABA(A) receptor agonists, and (4), whether gonadectomy decreases GAD(65 or 67) mRNA in steroid receptor-containing GABAergic neurons, and whether gonadectomy increases GABA(A)beta(3) receptor subunit mRNA expression in GnRH neurons. This research will further our understanding of the hypothalamic mechanism regulating tonic LH secretion in male and female rats, and the way in which negative, and perhaps positive, feedback steroids affect this mechanism.